The present invention relates to analytical methods. In a particular aspect, the present invention relates to methods for the identification of compounds which mediate the interaction between signal dependent transcription factors and co-factor protein(s) involved in the activation of transcription. In another aspect, the present invention relates to methods for the identification of new signal dependent transcription factors. In yet another aspect, the present invention relates to methods for the identification of novel co-factor protein(s) which mediate the interaction between signal dependent transcription factors and inducer molecules involved in the activation of transcription. In yet another aspect, the present invention relates to methods for treating diabetes mellitus.
Many eukaryotic genes are regulated in an inducible, cell type-specific fashion. Genes expressed in response to heat shock, steroid/thyroid hormones, phorbol esters, cyclic adenosine monophosphate (cAMP), growth factors. and heavy metal ions are examples of this class. The activity of cells is controlled by external signals that stimulate or inhibit intracellular events. The process by which an external signal is transmitted into and within a cell to elicit an intracellular response is referred to as signal transduction. Signal transduction is generally initiated by the interaction of extracellular factors (or inducer molecules, i.e., growth factors, hormones, adhesion molecules, neurotransmitters, and other mitogens) with receptors at the cell surface. Extracellular signals are transduced to the inner face of the cell membrane, where the cytoplasmic domains of receptor molecules contact intracellular targets. The initial receptor-target interactions stimulate a cascade of additional molecular interactions involving multiple intracellular pathways that disseminate the signal throughout the cell.
Many of the proteins involved in signal transduction contain multiple domains. Some of these domains have enzymatic activity and some of these domains are capable of binding to other cellular proteins, DNA regulatory elements, calcium, nucleotides, lipid mediators, and the like.
Protein-protein interactions are involved in all stages of the intracellular signal transduction processxe2x80x94at the cell membrane, where the signal is initiated in the cytoplasm by receptor recruitment of other cellular proteins, in the cytoplasm where the signals are disseminated to different cellular locations, and in the nucleus where proteins involved in transcriptional control congregate to turn on or turn off gene expression.
Mitogenic signaling affects the transcriptional activation of specific sets of genes and the inactivation of others. The nuclear effectors of gene activation are transcription factors that bind to DNA as homomeric or heteromeric dimers. Phosphorylation also modulates the function of transcription factors, as well. Oncogenes, first identified as the acute transforming genes transduced by retroviruses, are a group of dominantly acting genes. Such genes, which are involved in cell division, encode growth factors and their receptors, as well as second messengers and mitogenic nuclear proteins activated by growth factors.
The binding of growth factors to their respective receptors activates a cascade of intracellular pathways that regulate phospholipid metabolism, arachidonate metabolism, protein phosphorylation, calcium mobilization and transport, and transcriptional regulation. Specific phosphorylation events mediated by protein kinases and phosphatases modulate the activity of a variety of transcription factors within the cell. These signaling events can induce changes in cell shape, mobility, and adhesiveness, or stimulate DNA synthesis. Aberrations in these signal-induced events are associated with a variety of hyperproliferative diseases ranging from cancer to psoriasis.
The ability to repress intracellular signal-induced response pathways is an important mechanism in negative control of gene expression. Selective disruption of such pathways would allow the development of therapeutic agents capable of treating a variety of disease states related to improper activation and/or expression of specific transcription factors. For example, in patients with non-insulin dependent diabetes mellitus (NIDDM), hyperglycemia develops, in part as a result of xcex2 cell failure secondary to chronic insulin resistance. This hyperglycemia appears to be exacerbated by hyperglucogonemia and increased hepatic gluconeogenesis. cAMP appears to be the major starvation state signal which triggers glucagon gene expression as well as transcription of PEPCK, the rate limiting enzyme in gluconeogenesis.
There remains, thus, a need in the art for selective disruption of intracellular signal-induced response pathways.
In accordance with the present invention, it has been discovered that CREB binding protein (CBP) cooperates with upstream activators involved in the activation of transcription by signal dependent transcription factors, such as c-Jun (responsive to phorbol ester), serum response factor, and the like. Accordingly, assays employing CBP have been developed for the identification of compounds which disrupt the ability of signal dependent transcription factors to activate transcription. In another aspect, assays employing CBP have been developed for the identification of new signal dependent transcription factors. In yet another aspect of the present invention, assays employing CBP have been developed for the identification of novel co-factor protein(s) which mediate the interaction between signal dependent transcription factors and inducer molecules involved in the activation of transcription. In still another aspect, an assay is provided to identify compounds which have the binding and/or activation properties characteristic of CREB binding protein. In still another aspect, methods employing compounds which inhibit intracellular signal-induced response pathways have been developed for the treatment of diabetes mellitus.